1. Technical Field
The embodiments herein generally relate to enzymes of APases family. The embodiments herein more particularly relate to nucleic acid sequences coding for novel APases enzymes family.
2. Description of the Related Art
During the last two decades, APases including phytases have attracted considerable attention for both research and industrial applications in the areas of nutrition, environmental protection and health.
Monoesteric phosphatases (EC 3.1.3) commonly known as acid phosphatases (APases), catalyze the hydrolysis of phosphoric ester bonds of various substrates including phosphorylated sugars, lipids, proteins and nucleotides (Boyer et al., 1961). These enzymes are encoded by a highly diverse set of genes. Thaller and colleagues (1998) placed prokaryotic non-specific APases (NSAP) in three distantly related families A, B and C on the basis of shared conserved motifs despite of lack of overall sequence similarities. NSAPs are secreted enzymes which are produced as soluble periplasmic proteins or as membrane-bound lipoproteins, which are usually able to dephosphorylate a broad range of substrates and exhibit optimal catalytic activity at acidic to neutral pH values. Class A encompasses a group of bacterial APases which have a molecular mass around 25 kDa and carry a signature sequence motif defined as GSYPSGHT (SEQ ID NO: 9). Class B APases contain a polypeptide with a molecular mass of approximately 25 kDa for which FDIDDTVLFSSP (SEQ ID NO: 10) could be proposed as family motif sequence. Class C NSAP are a group with a molecular mass around 30 kDa and share four conserved aspartate residues. At the sequence level, class C enzyme appear to be related, although distantly, to class B and also to some plant acid phosphatases. Because of the presence of four invariant aspartate (D) residue within the most conserved domain among class B and C bacterial NSAPs and some plant APases, Rossolini and coworkers (1998) proposed a superfamily of DDDD (SEQ ID NO: 11) phosphohydrolyses.
Considering much higher sequence diversity in eukaryotic APases, Feizi and Malboobi classified plant APases into five distinct families with almost no similarities among them, even among the conserved family motifs. Considering the whole set of known APases in Arabidopsis thaliana and Oryza sativa as representatives of the dicotyledonous and monocotyledonous plants, the defined families were named as purple APase (PAP), Histidin APase (HAP), haloacid dehalogenase related APase ((HAD)-HRP), phospholipid APase (PLP) and SurE APase (SAP) families based on specific criteria and sequence similarities within them. These researchers proposed that the necessity for phosphate homeostasis for cellular survival has been the selective force which favored structural adaptations of various superfamily members toward APase activity to target as many alternative substrate types as possible. Then, divergent evolution within the families allowed broadening of substrate subtypes. For instance, these analogous families encompass four types of known phytase enzymes: HAP, PAP, cystein APase (CP) and a prokaryotic one named β-propeller phytase or BPP that are distinct both in terms of amino acid sequence and tertiary structure (Lung et al., 2008; Mullaney and Ullah 2005).
With respect to the important agricultural and industrial applications of APases, isolation of relevant genes has been of great interest and several gene isolation methods have been utilized.
A subset of these enzyme, named phytase, belongs to a special class of phosphomonoesterases [myo-inositol hexakisphosphate phosphorylase] and is capable of initiating the stepwise release of phosphate from phytate [myo-inositol (1, 2, 3, 4, 5, 6) hexakisphosphate], the major storage form of phosphate in plant (Greiner et al., 2002). For instance, phytases are now used as an animal feed additive to assist digestion of plant material for simple-stomached animals by liberating phosphate (Cromwell et al., 1995; Igbasan et al., 2001; Leesen et al., 2000; Simons et al., 1990; Miksch et al., 2002). The inorganic phosphate supplementation in the diets for simple-stomached animals can be reduced by including adequate amounts of phytase, and as a result, the fecal phosphate excretion of these animals can be reduced by as much as 50% (Arjula et al., 2009). Therefore, the utilization of phytase enzyme has been proposed as a means to reduce the level of phosphate pollution in the residuals of industries involving intensive animal production such as poultry or fish.
APases have a wide distribution in plants, microorganisms and also in some animal tissues (Greiner et al., 1993; Dvorakova 1998; Konietzny and Greiner 2002). Recent research has shown that microbial APases are the most promising ones for biotechnological application in terms of cost, ease of production and processing (Pandey et al., 2001). APases have been detected in various bacteria, such as Bacillus sp. (Choi et al., 2001; Kerovuo et al., 1998; Kim et al., 1998; Shimizu 1992), Pseudomonas sp. (Irving and Cosgrove 1971; Richardson and Hadobas 1997), Pseudomonas syringae (Cho et al. 2003), Escherichia coli (Golovan et al. 2000; Greiner et al. 1993), Enterobacter (Yoon et al., 1996), Klebsiella sp. (Greiner et al., 1997), Citrobacter braakii (Kim et al., 2003), Lactobacillus sanfranciscensis (De Angelis et al. 2003), Pantoea agglomerans (Greiner 2004) and Pseudomonas putida (Malboobi et al., 2009). Also, several bacterial phytase-encoding genes have been cloned from Bacillus sp. (Kim et al., 1998), Escherichia coli (Rodriquez et al., 1999; Golovan et al., 2000), Klebsiella sp. (Sajidan et al., 2004), Obesumbacterium proteus (Zinin et al., 2004), Pseudomonas syringae (Cho et al., 2005), Yersinia intermedia (Huang et al., 2006), and Citrobacter sp. (Luo et al., 2007). For lactic acid bacteria, however, the results were inconsistent; a few strains seem to have a quite low phytase activity, while for the majority of strains no phytase activity was detected. Recently it was shown that lactic acid bacteria isolated from sourdoughs exhibited a considerable phytate degrading capacity (De Angelis et al., 2003). Among the different lactic acid bacterial strains isolated from sourdoughs, Lactobacillus sanfranciscensis, which is considered as a key sourdough lactic acid bacterium, was identified as the best phytase producer. The APases produced by fungi are extracellular, whereas the enzymes from bacteria are mostly cell associated. The only bacteria showing extracellular phytase activity are those of the genera Bacillus and Enterobacter. The APases of Escherichia coli have been reported to be periplasmatic enzymes and phytase activity in Selenomonas ruminantium and Mitsuokella multiacidus was found to be associated with the outer membrane (D'Silva et al., 2000).
Apart from fungi and bacteria, APases including phytase have been isolated and characterized from cereals such as triticale, wheat, maize, barley and rice and from beans such as navy beans, mung beans, dwarf beans and California small white beans that generally have lower enzyme activities than the bacterial ones. In general, legumes and oilseeds exhibit a 10-fold lower activity compared to cereals (Vohra and Satyanarayana 2003; Konietzny and Greiner 2002).
Since certain APases have preferred substrate ranges (Shamsuddin 2002, Vucenik et al., 2003, Oh et al., 2004), APases may find biotechnological applications in food processing to improve meal quality in particular for the reduction of phytate contents in feed and food (Lei et al., 2001; Vohra and Satyanarayana 2003; Haefner et al., 2005), in diagnostic kits as an stable, strong indicator enzyme and in mining industry as bioleaching agent. Depending on the application, an APase in which there is commercial interest, certain criteria should be met. Enzymes used as feed additives should be effective in releasing phosphates from phytate in the digestive tract, stable to resist inactivation by heat from feed processing and storage, and cost-effective for production. Thermo stability is a particularly important issue since feed pelleting is commonly performed at temperatures between 65° C. and 95° C. Although an after-spray apparatus for pelleted diets and/or chemical coating of phytase may help by passing the hot steps, thermostable phytases are still better candidates for feed supplements (Arjula et al., 2009).
So far naturally occurring APases having the required level of thermo stability for application in animal feed have not been found in nature (Lei et al., 2001). Up till now, two main types of APases have been identified; acid APases with an optimum pH around 5.0 and alkaline APases with an optimum pH around 8.0 (Oh et al., 2004). Most of the so far described microbial APases belong to the acidic ones and their pH optima range from 4.0 to 5.5.
Due to the shortage in nonrenewable resources of phosphorus, costs of production and environmental pollution concerns, there is a great desire to utilize APases, particularly in the area of food and feed production. Such enzymes must possess certain criteria for industrial applications such as high specific activity, thermo stability and activity in a broad range of pH. Hence there is a need for a cost effective and competitive production of APases with high yield, high specific activity and required purity level for desired industrial applications.
The above mentioned shortcomings, disadvantages and problems are addressed herein and which will be understood by reading and studying the following specification.